Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.2 Eye Color Mutant ScreeningPage 1 of 23.2 Eye Color Mutant ScreeningMark BenedictIntroductionAnopheles mosquitoes undergo induced color change (called homochromy) based on perception of thebackground against which they are cultured. 1 When larvae are reared on either a dark black or whitebackground, they become pigmented dark or pale respectively as shown by the pair of An. albimanuslarvae in Figure 3.2.1. The degree of darkening depends in part on the length of time the larvae havebeen cultured in a black container and the degree of fat body development. Therefore, larvae culturedduring their entire development in a dark container at a low density show this change most dramatically.This color change depends on the normal eye pigmentation and, presumably, on the proper function ofany pigmentation and signaling pathways involved in the response.The method simply requires culturing the larvae from at least the second stage in black or dark-coloredcontainers that are illuminated. (Larvae cultured in darkness will develop typical pale pigmentation.) Thesource of illumination does not appear to be critical. Occasional transfers of a few minutes to whitecontainers for feeding or correcting the density does not interfere with the effect.At the L3 or L4 stage, larvae are scanned en masse in the dark tray in a well-illuminated location for thosethat appear lighter in color. They are usually quite apparent as demonstrated by the two larvae in Figure3.2.2, but purposely seeding a sample of dark larvae with a few that are pale will demonstrate the degreeof effect that can be expected. These individuals are transferred to a dish for microscopic examination.Leaving the larvae undisturbed during examination provides better visualization since the dorsal sidecoloration is a more consistent indicator of general color. After this initial selection, it is also helpful totransfer the larvae to a white tray and scanning for pale individuals. Usually, no more than approximately25 larvae per thousand cultured in this way require individual examination.Figure 3.2.1. Anopheles albimanus larvae rearedon a black background (top) and a whitebackground (bottom).Figure 3.2.2. Eye color mutants are easier to detecton a dark background as they will not changepigmentation and will appear much lighter in color.1 Before beginning a large screen, it is advisable to culture a thousand or so larvae of the species ofchoice in dark containers. While all individuals of most species change color, some laboratory stockshave a low frequency of individuals that do not.